Name:_____________________________________ Date:________________________

Year and Section:___________________________ Instructor:___________________

MODULE 6

The Nucleus of the Cell

I. OBJECTIVES

At the end of this module, you should be able

• To demonstrate a clear understanding of the structure, functions and processes inside a

cell’s nucleus
• To identify and describe the parts of a cell nucleus;
• To describe the molecular structure of genes and their organization to chromosomes;
• To explain thoroughly the events and processes that happen in cell cycle;
• To identify mechanisms that drive nuclear genome evolution; and
• To discuss the basic concepts of prokaryotic and eukaryotic gene expression

II. COURSE MATERIAL

Anatomy of the Cell Nucleus

The nucleus is an organelle found in eukaryotic cells that serves as information processing and
administrative center of the cell. The nucleus has two main functions: (1) to store the genetic
material and (2) to coordinate cellular activities such as growth, metabolism, protein synthesis
and reproduction. The nucleus has the following parts:

TASK: Given the following description of the parts of the nucleus, identify the
structures in the figure. Put the letter in the box provided.
a) Nuclear envelope - a double membrane
that encloses the content of the nucleus The nucleus
and separate it from the contents of the
cytoplasm.
b) Nuclear lamina – composed of lamins that
lines the inner surface of the nuclear
envelope. It provides structural support to
the nuclear membrane.
c) Nuclear pore – regulates the transport of
specific molecules inside and outside the
nucleus. It is also connected to the
endoplasmic reticulum where protein
synthesis occurs.
d) Nucleoplasm – the semifluid matrix found
inside the nucleus where nucleotides and
enzymes are found.
e) Chromatin – dense string-like structures
composed of nucleic acids associated with
proteins (also referred to as chromosome
territories)
f) Nucleolus – dark staining region within the nucleus responsible for the synthesis of ribosomal
RNA

MOLECULAR STRUCTURE OF GENES AND CHROMOSOMES

Structure of Gene

By the beginning of the twenty first century, molecular biologists had completed sequencing the
entire genomes of hundreds of viruses, bacteria, unicellular eukaryotes, plants and even humans.
These efforts led to the extensive elucidation of the structure of the fundamental units of
inheritance – the gene.

In molecular biology the term gene refers to the entire DNA sequence that is needed for the
synthesis of a functional protein. However, there’s more to gene structure than just the coding
region (exons). In order for a gene to be complete, such that the coding sequence will be
expressed, several sequences must be present:

• Promoter - It acts as a 'genetic dimmer switch' in that it turns the gene on and off and specifies
how many copies of the protein will be produced. The promoter allows the binding of
transcription factors and recruit RNA polymerase;
• Enhancer – These are regulatory sequences that enhance the transcription of gene;
• Termination Sequence – This is the last region and signals the end of the gene and its
transcription.

In prokaryotes and some viruses, genes coding for functionally related proteins are group in a
single cluster called operons, while eukaryotic genes are divided by long intergenic non coding
sequences called introns.

Figure 2. The basic structure of a gene comprises of the promoter, the coding region, the
termination sequence and the enhancer.
Chromosome Organization

Inside the a eukayotic cell’s nucleus, the DNA molecule is packed into thread like structures called
chromosomes. For instance, the DNA from all the 46 human chromosomes measures
approximately 2 meters when laid out. Considering the size of human cell of about 10 um, the
DNA must be tightly packaged to fit inside the cells’ nucleus. At the same time, it must be readily
accessible for the genes to be expressed.

There are a number of ways by which the chromosomes are compacted. During interphase (the
stage where cells are not dividing), the genetic material exists as a nucleoprotein complex called
chromatin. The most common proteins
associated with DNA are the histones.
Short stretches of DNA double helix wraps
around around the core of eight histone
proteins forming a “beads on a string”
structure. Histone proteins provide
structural support to DNA, giving the
chromosome a more compact shape. This
histone-DNA complex is called a
nucleosome. In a nucleosome unit, a 147
base pair DNA is wrapped around a histone
core octamer composed of H32 - H42
tetramer associated with two H2A – H2B
dimer. Approximately, a DNA molecule in
this form is seven times shorter than the
double helix without the histones. As the
nucleosomes and the linker DNA between
them are coiled into a 30 nm fiber by the
action of H1 histone, the chromosomes are
50 times shorter than its extended form. In
this condensed state, nucleosomes are
thought to be packed into irregular spiral or
solenoid arrangment.

Further condensation and coiling of the

chromatin structure is facilated by a variety
of fibrous proteins or scaffold proteins.
Scaffold proteins are non-histone proteins
that produce highly condensed structure
for the chromosomes. These fibrous proteins also ensure that chromosomes occupies specific
areas in the nucleus of a non- dividing cell such that chromosomes does not over lap with each
other. This series of condensation and compaction results to a DNA molecule packed to a
chromosome that is 10,000 fold shorter that its extended form. The process of chromosome
organization is illustrated in the figure.

Often, a chromosome is depicted as an X structure with

a constriction along its length that divides the
chromosome into two sections, the long arm and the
short arm. The constriction is called the centromere. It
is the site where spindle fibers attach. During cell
division, the centromere is genetically active while the
rest of the chromosome parts are inactive.

Associated with the centromere is the kinetochore,

which is a complex of proteins where the microtubules
of the soindle attache dutring cell division. Normally,
each diploid homologous chromosome exhibits
additional constirctions called secondary constrictions.
These constrictions are mainly responsible for the
formation of the nucleolus. The tips or the terminal ends
of the chromosome are called telomeres. The telomeres has a property that protects the
chromosome from detoriation and fusion with other chromosomes

Genome Packaging in Prokaryotes.

Prokaryotes do not contain nuclei or any membrane bound organelles. The nucleoid is simply
the area where the double stranded circular chromosomal DNA is located. Prokaryotic genome
is usually larger than the cell itself. The huge chromosome of prokaryotes is packed inside the
cell through supercoiling. Supercoiling is analogous in twisting a rubber band to make tiny coils
and twisting it again to make even smaller coils. Prokaryotic genomes can either be negatively
supercoiled, when the DNA is twisted in a direction opposite to that of the double helix, or
positively supercoiled when the DNA is twisted in the same direction as the double helix.

Whereas in eukaryotes, DNA wraps around histone proteins, prokaryotes do not have histones.
Instead multiple non-histone proteins act together to fold and condense the prokaryotic DNA. For
instance, HU proteins are very abundant in the nucleoid and work with DNA topoisomerase I to
introduce sharp bends in the chromosome necessary to generate negative supercoils. Moreover,
IHFs (integration host factors) bind within the genome to produce additional bends. Once
condensed, enzymes like DNA topoisomerase I and DNA Gyrase maintain the supercoil.

Although at compacted state, the prokaryotic genome is still accessible for transcription factors.
Because there is nuclear membrane that separates the DNA from the ribosomes in the cytoplasm,
transcription and translation happen simultaneously in these organisms.

Exercise. Compare and contrast eukaryotic and prokaryotic genomes. Focus on their size,
localization, and organization.
Prokaryotic Eukaryotic

THE CELL CYCLE

Overview of the Cell Cycle

The only way to make a new cell is to duplicate a cell that already exist. All living organisms, from
unicellular bacterium to the multicellular mammals are products of repeated cell growth and
division. A cell reproduces by performing an orderly sequence of events in which it duplicates its
contents and divides in two. This series of duplication and division is known as cell cycle. Cell
cycle is an essential mechanism by which all living organisms reproduce. The cell, through the
cell cycle, accomplishes its most fundamental task : the passing on of its genetic information to
the next generation of cells.

The details of cell cycle vary from species to species at different times in an organism’s life.
However, certain features are universal. The cell cycle is a 4-stage process composed of the non-
dividing phase (intephase) and the dividing phase (mitotic phase).
Interphase

Most of the cells require much more time to grow and

double their mass of proteins and organelles than they
require in replicating their chromosomes and divide. Partly,
to provide time for growth, cell cycles have gap phases.
The first gap called G1 phase occurs after cell division and
before DNA replication phase. This is often referred to as
the growth phase because during this stage the cell
continues to grow accompanied by the synthesis of
enzymes and proteins for DNA replication and cell division.
The cell is fully functional and the centromere and the
components of centrosome are made during this phase.
Lastly, the G1 phase ensures that the cell is biologically
ready before it undergoes DNA replication.

The G1 phase is followed by the S-phase (S for DNA

synthesis). As discussed, the most basic function of cell cycle is to duplicate the vast amount of
DNA in the chromosomes and to segragate the copies to the two daughter cells. Chromosome
duplication occurs at S-phase and requires 10-12 hours, thus occupying the half of the cycle in a
typical mammalian cell. At the end of the S phase, the DNA molecules in each duplicated
chromosomes are intertwined and held tightly by specialized protein linkages.

The S-phase is followed by another intermediary phase called G2 phase. This gap phase acts as
a safety gap where the cell ensures that the entirety of its DNA and other cellular components are
duplicated before it proceeds to division. At this stage, the centrioles are fully formed and other
microtubules necessary to mobilize the chromosomes during cell division are assembled. This
gap phase is the last chance for the cell to grow before it splits ino two independent cells during
cell division.

The G1 phase, S phase and G2 phase comprise the non-dividing stage of the cell cycle called the
interphase.

M Phase

Following the completion of S phase and transition through G2, the cell undergoes the M phase.
This begins with mitosis, in which the sister chromatids are separated and segragated to a pair of
identical daughter nuclei, each having its own copy of the genome. Mitosis is divided into our
stages – prophase, metaphase, anaphase and telophase – each defined baased on the behavior
of the chromsomes as observed under the microscope.

As mitosis is completed, the second major event of cell division proceeds – cytokinesis in which
the cell is divide into two halves eah with identical nucleus. Panel 1 describes the events that
occur during mitosis.
 Exercise. The lung epithelial cells of newt undergoing the M phase were shown. Order
these micrographs and identify the stages of M phase shown.

ANSWERS:

Stages of Cell Division

Regulation of the Cell Cycle

Cell move through the cell cycle in a regulated manner. They use internal cues and environmental
signals to decide whether to proceed to cell division or not. This regulation ensures that cells will
divide only under favorable conditions. For instance, DNA damage and lack of space in the tisseu
or organ may cease the progression of the cell to divide. Cell cycle is mainly regulated through
cell cycle checkpoints. A checkpoint is a surveillance mechanism that monitor the integrity,
fidelity and order of the major events in the cell cycle. Chekcpoints play critical roles in preventing
the cells from progressing to the next phase of the cell when the prior phase has not been
completed. Premature entry to phases of the cell cycle can lead to catastrophic consequences
for the cells such as cell death. There are a lot of checkpoints in eukaryotic cell cycle. The three
major ones are the following:
1. G1 Checkpoint (G1 / S transition)

It is the first chekcpoint located at the end og G1 phase and just before the start of the S phase.
It is where the cell deicdes whether to divide, to delay division or to enter a resting stage. At this
checkpoint a cell must ensure that its size, the availability of nutrients, the presence of signals
and DNA damage are assessed. If the cell does not get the “go cues” from G1 checkpoint, it may
leave the cycle and enter a resting stage called G0 . Some cells stay at the G0 for their entire
lifetime while other resume the cycle once theenvironmental conditions become favorable for
them to divide. Once the cell passes the G1 checkpoint and enters the S phase, it will make an
irreversible commitment to divide, barring problems such as errors in replication and DNA
damage.

2. G2 Checkpoint (G2 / M transition)

This is the check point before the cell enters the division phase. The G2 checkpoint prevents
initiation of mitosis when DNA is damaged. If the chekpoint detects DNA damage, the cell cycle
is halted and the cell either completes the replication or repair DNA damage. When the the
damage is irreparable, the cell will proceed to programmed cell death or apoptosis to ensure that
damaged DNA is not passed on to the daughter cells. The G2 checkpoint is also an imprtant focus
in understanding the molecular biology of cancer.

3. Spindle Assembly Checkpoint (SAC, metaphase/anaphase transition)

This checkpoint ensures that each of the sister chromatid iscorrectly attached to the spindle
microtubule via the kinetochore. The SAC acts to maintain genome stability by delaying cell
division until accurate chromosome segragation is guaranteed.

The cell cycle checkpoints work through the cascades of internal and external cues that trigger
signalling pathways involve in the activation and inactivation of core proteins that moves the cell
cycle forward. These sets of proteins are called cell cycle regulators.

• Cyclins

These are regulatory proteins that control progression of the cell cycle. Each cyclin is
associated with the phase, transition or set of phases. Cyclins help drive the major events
of eachphase of the cell cycle. For instance, M –cyclins promotes events in M phase such
as nuclear membrane digestion and chromosome condensation.

• Cyclin dependent kinases (CdKs)

Cyclins activate and deactivate many proteins inside the cel such as the cyclin dependent
kinases (CdKs). CdKs are kinases that phosphorylates specific target proteins. The
attached phopshate makes activates or deactvates the target protein. For instance, G1/S
cyclins sends CdKs to S phase protein promoting cell division. A lone CdK is inactive, but
the binding of cyclin activates it to modify specific traget proteins. Cyclin/CdK complex
phosphorylates target proteins need for the progression of the cell cyle.
 • Maturation Promotion Factor (MPF)

MPFs add phosphate tags to several proteins in the nuclear envelop and signals its
breakdown. MPF also induces chromosome condensation, a key event that initiates M
phase.

• Anaphase-promoting complex / cyclosome (APC/C)

APC/C is a protein complex that breaks down M cyclins at the beginning of anaphase.
Aside from this, APC/C mobilizes the sister chromatids during anaphase by breaking down
the proteins that hold the sister chromatids together. The degradation of these proteins
pushes the cells out of mitosis and allows the daughter cells to enter the G1 phase. APC/C
performs this task by adding small protein tags called ubiquitin (Ub) to its target proteins.
Proteins tagged with ubiquitin are send to proteosome and are degraded.

Cell cycle checkpoints and regulators work together to ensure the successful completion of the
cell cycle. So, how do checkpoints and regulators perform their task? As an example, let us look
at how DNA damage halts cell cycle in G1 phase.

DNA damage is brought about by exposure to

UV rays, ingestion of mutagens and many other
instances. Cells deal with this damage by
utilizing the key response protein called p53, a
known tumor suppresor.

The p53 protein arrests the cell cycle in G2 by

trigerring the production of CdK inhibitors (CKI)
CdK inhibitors bind to CdK-cyclin complexes and
block their activities. Then, p53 activates a DNA
repair mechanisms. Lastly, if the damage is
reparable, the p53 protein triggers programmed
cell death. This ensures that the damage genetic
material will not be passed on to the daughter The interaction of cell cycle checkpoint and
regulators ensures the completion of the cell
cells. cycle.
 Exercise. Checkpoint ensures successful progression of the cell cycle. Fill out the
table with necessary key points for each cell cycle checkpoint. Identify at least two for
each.
Checkpoint Checks for?

G1 Checkpoint

G2 Checkpoint

M checkpoint

NUCLEAR GENOME EVOLUTION

Genome evolution is the process wherein the genome changes in structure and size over time.
Genome evolution has several mechanisms that allow the genes and genomes to evolve and
produce the vast diversity of modern life form on earth.

The following are the common mechanisms of nuclear genome evolution:

1. Mutation results addition or deletion of one or more nucleotide bases causing changes in the
reading frame, hence the entire code will be read in a different order from the original resulting
to non-functional proteins.

2. In gene duplication, the coding region of a gene is duplicated. This process can either be
due to errors in recombination or through retrotransposition. Duplicated genes are thought to
be immune in selection pressure, hence may accumulate large amounts of mutations.

3. Similar to gene duplication, whole genome duplication is the process in which the organism’s
entire genome is copied. This event is the result of non-disjuction during meiosis. Non
disjuction refers to the failure of chromosomes to segregate during cell division resulting to
imbalance in chromosome numbers. Often, whole genome duplication leads to polyploidy.

4. Transposons are genetic elements called jumping genes as they can insert themselves in a
different location along the genome. Transoposons works in two mechanisms. The first one
is where a certain portion of the genome is excised and transferrred to another region of the
genome (cut and paste). The second mechanisms beigns with making multiple copies of a
certain part if genome and transfer those copies to another location in the genome (copy and
paste).

5. Exon shuffling refers to the process by which two or more exons from different genes were
brought together, or the same exon is dupicated resulting to a new exon-intron structure. This
is a mechanism for creating new genes.
6. Horizontal Transfer happens when a piece of DNA is transferred from the genome of one cell
to the that of the other. It is incontrat to the vertical gene transfer which happens from parents
to progenies.

Exercise. Identify the mechanism of genome evolution depicted in the following figures. Put your
answer in the box provided.
Questions:

1. What is the role of nucleus in a eukaryotic cell?

2. Kinetochores are proteins associated with centromeres. How many kinetochores are present
in a human cell as it enters mitosis?
3. Cancer is a disease primarily characterized by development of abnormal cells that divide
uncontrollably. Which phase(s) of the cell cycle is affected by cancer?
4. Among the mechanisms of genome evolution, which do you think generates the most diversity
among life forms? Discuss your answer.